Rapid construction of parallel analysis of RNA end (PARE) libraries for Illumina sequencing

Methods 67 (2014) 84–90

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Rapid construction of parallel analysis of RNA end (PARE) libraries for Illumina sequencing Jixian Zhai a,b,1, Siwaret Arikit a,b,1, Stacey A. Simon a, Bruce F. Kingham b, Blake C. Meyers a,b,⇑ a b

Department of Plant & Soil Sciences, University of Delaware, Newark, DE 19711, USA Delaware Biotechnology Institute, University of Delaware, Newark, DE 19711, USA

a r t i c l e

i n f o

Article history: Available online 28 June 2013 Keywords: miRNA PARE RNA Degradome Illumina sequencing

a b s t r a c t MicroRNAs (miRNAs) are 21 nt small RNAs that pair to their target mRNAs and in many cases trigger cleavage, particularly in plants. Although many computational tools can predict miRNA:mRNA interactions, it remains critical to validate cleavage events, due to miRNA function in translational repression or due to high rates of false positives (over 90%) for unvalidated target predictions. A few years ago, three laboratories described similar methods to validate cleavage of miRNA targets by the cloning en masse of 50 ends of cleaved or uncapped mRNAs. To take advantage of the recent progress in high-throughput sequencing technology, we have devised an updated protocol to (1) enable much faster library preparation, and (2) reduce the cost by pooling indexed samples together for sequencing. Here we provide a step-by-step protocol for PARE library construction, starting from total RNA. This protocol has been successfully used in our laboratory to validate miRNA targets in a variety of plant species. We also provide advice for troubleshooting on some common issues. Ó 2013 Elsevier Inc. All rights reserved.

1. Introduction In plants, miRNAs function primarily through the cleavage of their target messenger RNAs (mRNAs) that have near-perfect complementarity at their target sites [1]. Therefore, computational approaches can be applied to perform scans on either genome or cDNA for potential targets based on the base pairing, and this pairing can be further scored by proposed miRNA: target interaction rules [2–4]. However, the false positive rate of the predictions remains high because targeting involves many other factors; for example, different spatiotemporal expression patterns may mean that good miRNA–mRNA homologies do not result in cleavage. Therefore it is necessary to experimentally validate the predicted targets. This may be done using a modified version of 50 RACE (Rapid Amplification of cDNA Ends) that can clone and map by sequencing a specific cleavage site. In 2008, three labs published and differentially named techniques based on high-throughput sequencing to validate miRNA targets, including PARE (parallel analysis of RNA ends) [5], degradome sequencing [6], and GMUCT (genome-wide mapping of uncapped and cleaved transcripts) [7]. These approaches all took advantage of the uncapped 50 end of cleaved mRNA 30 end products, capturing this sequence via ligation with an RNA adapter. ⇑ Corresponding author at: Department of Plant & Soil Sciences, University of Delaware, Newark, DE 19711, USA. Fax: +1 302 831 4841. E-mail address: [email protected] (B.C. Meyers). 1 These authors contributed equally to this work. 1046-2023/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ymeth.2013.06.025

Both PARE and the degradome technique generate an isolength library (consistently sized fragments) via digestion by MmeI to isolate a 20-nt piece or ‘‘signature’’ from the 50 end of the doublestranded cDNA [5,6]. A dsDNA 30 adapter was ligated to the MmeI-digested products and then the resulting fragment was PCR amplified, gel purified, and sequenced. We and collaborators have previously published a protocol for PARE library construction that required approximately 1 week to prepare and utilized the older Illumina GA sequencing [8]. That protocol also requires many gel purification steps and the samples are difficult to pool for sequencing. Here, we describe an improved protocol for the library construction that reduces the total time to 3 days, uses only two gel purification steps, and the samples can be easily pooled for sequencing to benefit from the enormous sequencing depth of the Illumina Hi-Seq technologies (Fig. 1). 2. Materials 2.1. Reagents DynabeadsÒ mRNA Purification Kit (610-06), Invitrogen AgencourtÒ AMPureÒ XP, 60 ml (A63881), Beckman-Coulter Primers from TruSeqÒ Small RNA Sample Prep Kit-Set A (RS-200-0012), Illumina T4 DNA Ligase 100,000 units (M0202M), NEB T4 RNA Ligase (AM2141), Invitrogen MmeI (R0637S), NEB

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RNaseOUT™ Recombinant Ribonuclease Inhibitor (10777-019), Invitrogen Acrylamide/Bis 19:1 40% (w/v) Solution (2  500 ml bottles) (AM9024), Invitrogen High-Sensitivity DNA Kit (5067-4626), Agilent Technologies Quant-iT dsDNA HS Assay Kit (Q32851), Invitrogen TruSeq SR Cluster Kit V3 (GD-401-3001), Illumina Blue/Orange Loading Dye, 6 (G1881), referred as 6 GLB (Gel Loading Buffer) Ethidium bromide (PRH5041), Fisher DEPC-Treated Water (AM9906), Invitrogen Molecular Biology Grade Ethanol, 500 ml (BP2818500), Fisher 2.2. Equipment Mini-PROTEAN Tetra Cell 4-gel vertical electrophoresis system (165-8001), Biorad MagneSphereÒ Technology Magnetic Separation Stands (Z5342), Promega Qubit Fluorometer (Q32866), Invitrogen Bioanalyzer (G2938C), Agilent Technologies cBot Cluster Generation System, Illumina 2.3. Adapters and sequencing primer 50 -PARE RNA adapter: 50 -GUU CAG AGU UCU ACA GUC CGA C-30 Target RT-primer: 50 -CGA GCA CAG AAT TAA TAC GAC TTT TTT TTT TTT TTT TTT-30 50 cDNA PCR primer: 50 -GTT CAG AGT TCT ACA GTC CGA C-30 30 cDNA PCR primer: 50 -CGA GCA CAG AAT TAA TAC GAC T-30 dsDNA_top: 50 -TGG AAT TCT CGG GTG CCA AGG-30 (PAGE purified) dsDNA_bottom: 50 -CCT TGG CAC CCG AGA ATT CCA NN-30 (PAGE purified) Final PCR primer: 50 -AAT GAT ACG GCG ACC ACC GAC AGG TTC AGA GTT CTA CAG TCC GA-30 Indexed TruSeq 30 PCR primers, Index 1–24 PARE sequencing primer for Illumina HiSeq: 50 -CCA CCG ACA GGT TCA GAG TTC TAC AGT CCG AC-30 3. Procedure 3.1. Day 1

Fig. 1. Schematic depiction of PARE library construction. The PARE library made from this protocol is suitable for identification and validation of miRNA targets; also, more than one PARE library can be pooled and sequenced together. The procedure includes: (1) polyadenylated RNA isolation; (2) 50 -RNA adapter ligation to the 50 -ends (50 -monophosphate) of the single-stranded, cleaved RNA molecules; (3) reverse transcription, generating the first strand of cDNA using an oligo(dT) with a 30 -adapter sequence; (4) second strand synthesis and PCR amplification; (5) MmeI digestion to generate signature fragments (20 bp downstream); (6) ligation of MmeI digested products with a double-stranded 30 -DNA adapter with degenerate nucleotides in the overhanging region; (7) PCR amplification of PAGE-purified products ligated to the 30 -DNA adapter (step not shown), and PAGE purification of the final product (step not shown); (8) library pooling (up to 8 samples); (9) library sequencing via Illumina technology using a HiSeq instrument. Note that individual PARE libraries can be filtered after the sequencing by the index sequences on the 30 indexed adapters (show in different colors).

PHUSION HOT START II polymerase 100 U (F549), Fisher Corning Costar Spin-x column (07-200-387), Fisher 25 bp DNA ladder (G4511), Promega 10 bp DNA ladder (G4471), Promega GlycoBlue™ (15 mg/ml) (AM9516), Invitrogen SuperScriptÒ III Reverse Transcriptase (18080044), Invitrogen

3.1.1. Purify poly(A) RNA using Invitrogen Dynabeads mRNA Purification Kit (20 min) RNA quality and integrity, which is critical to the success of the PARE library construction, should be checked prior to the following preparation.  Dynabeads/Binding Buffer suspension preparation: Transfer 200 ll of well resuspended Dynabeads to a 1.5 ml microcentrifuge tube. Place the tube on the magnetic stand for 30 s or until Dynabeads have migrated to the tube wall. Pipette off the supernatant. Remove the tube from the stand, add 100 ll Binding Buffer to calibrate beads. Put the tube back on the magnetic stand. Pipette off the supernatant, then remove the tube from the stand. Add another 100 ll Binding Buffer to the Dynabeads (optimal hybridization condition is a 1:1 ratio, relative to sample volume).  Starting RNA preparation: Use 75 lg of total RNA as starting material (the amount can be as low as 40 lg). Adjust the volume of the 75 lg total RNA to 100 ll with DEPC-treated water. Heat to 65 °C for 2 min to disrupt secondary structures, then place on ice.

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 Poly(A) RNA isolation: Add the total RNA to the Dynabeads/Binding Buffer suspension. Mix thoroughly, rotate on a roller or mixer for 5 min at RT to allow the poly(A) RNA to anneal to the oligo (dT)25 on the beads. Place the tube on the magnetic stand until the solution is clear. Pipette off the supernatant. Remove the tube from the stand and wash the poly(A) RNA-bead complex TWICE with 200 ll Washing Buffer B (remove all the supernatant between each washing step with the help of the magnetic stand). Elute the poly(A) RNA from the beads by adding 16 ll of Elution Buffer (10 mM Tris–HCl, pH 7.5). Heat the tube to 65 °C for 2 min and then place immediately on the magnetic stand. Transfer 15 ll of the eluted poly(A) RNA to a new tube. 3.1.2. Ligation of the 50 adapter (1 h) Prepare 20 ll ligation reaction according to the below formula. Poly(A) RNA 50 RNA adapter (200 lM) 10 RNA Ligase Buffer T4 RNA Ligase RNase OUT TOTAL

15 ll 1 ll 2 ll 1.5 ll 0.5 ll 20 ll

Incubate the ligation reaction at 37 °C for 50 min. Remove the reaction from 37 °C and add 80 ll of DEPC-treated water to adjust the volume to 100 ll. Terminate the ligation reaction by heating at 65 °C for 10 min, then place on ice. 3.1.3. 50 Adapter Ligated poly(A) RNA Purification using Dynabeads to remove Unligated 50 adapters‘ (20 min)  Dynabeads/Binding Buffer suspension preparation: Prepare the Dynabeads suspension in the same way as described above.  50 -Adapter Ligated poly(A) RNA Purification: Add the 100 ll ligation reaction to the Dynabeads/Binding Buffer suspension. Mix thoroughly, rotate on a roller or mixer for 5 min at RT to allow the poly(A) RNA to anneal to the oligo (dT)25 on the beads. Place the tube on the magnetic stand until the solution is clear. Pipette off the supernatant. Remove the tube from the stand and wash the poly(A) RNA-bead complex TWICE with 200 ll Washing Buffer B (remove all the supernatant between each washing step with the help of the magnetic stand). Elute the poly(A) RNA from the beads by adding 28.5 ll of Elution Buffer (10 mM Tris–HCl, pH 7.5). Place the tube at 65 °C for 2 min and then place immediately on the magnetic stand. Transfer 27.5 ll of the eluted poly(A) RNA to a new PCR tube. 3.1.4. Reverse transcription and double-stranded cDNA synthesis (90 min) Denaturation Prepare the reaction as following: 50 Adapter ligated RNA RT Primer (100 lM) TOTAL

Denature at 65 °C for 5 min, then cool at RT 1st strand cDNA synthesis

27.5 ll 2 ll 29.5 ll

RT RNA 5 1st strand Buffer 10 mM (each) dNTP mix 0.1 M DTT RNase OUT Superscript III TOTAL

29.5 ll 10 ll 2 ll 2.5 ll 2 ll 4 ll 50 ll

On PCR machine: 42 °C for 50 min, 72 °C for 10 min, 4 °C HOLD 2nd strand cDNA synthesis (cDNA PCR) 1st strand cDNA 100% DMSO 5 GC Buffer 10 mM (each) dNTP mix 50 cDNA primer (10 lM) 30 cDNA primer (10 lM) dH2O Phusion polymerase TOTAL

20.0 ll 1.5 ll 10 ll 1.25 ll 2.0 ll 2.0 ll 12.25 ll 1 ll 50 ll

PCR: 98 °C for 1 min, (98 °C for 30 s, 58 °C for 30 s, 72 °C for 5 min) 7 cycles total, 72 °C for 7 min, 4 °C HOLD 3.1.5. Purify PCR amplified cDNA using Agencourt AMPure XP PCR Purification System (15 min)  Add 90 ll of AMPure XP beads to 50 ll of PCR amplified cDNA in a 1.5 ml microcentrifuge tube. Mix thoroughly by pipetting gently up and down 10 times. Incubate the mixed samples for 5 min at RT. Place the tube on the magnetic stand for 2 min or until beads have migrated to the tube wall. Aspirate off the supernatant and discard. While on the magnetic stand, add 200 ll of freshly prepared 70% EtOH. Let sit for 30 s at RT. Aspirate off the EtOH and discard. Repeat for a total of two washes. Dry the pellet on the stand for 5–7 min. Remove the tube from the stand. Add 16 ll of dH2O. Pipette to resuspend. Place the tube back on the stand for at least 1 min to separate the beads from the PCR product. 3.1.6. MmeI Digestion (1 h) Purified PCR product 10 NEB4 Buffer SAM (32 mM) MmeI (2 u/ll) TOTAL

15.8 ll 2 ll 0.2 ll 2 ll 20 ll

37 °C (50 min); remove from 37 °C; heat kill at 65 °C for 10 min; Let cool at RT (do NOT place on ice). Prepare dsDNA adapter duringdigestion. 3.1.7. 30 -Double-strand DNA adapter ligation (90 min) Make freshly prepared duplex adapter by annealing oligos dsDNA_top (100 lM) dsDNA_bottom (100 lM) TOTAL

25 ll 25 ll 50 ll

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100 °C (5 min); slow cool down to RT by stepping temp down 5 °C every 30 s (0.1 °C/s to 25 °C). Keep at RT. Set up duplexligation: MmeI digested PCR Annealed duplex adapter (100 lM) 10 T4 DNA Ligase Buffer T4 DNA Ligase (2000 u/ll) dH2O TOTAL

20 ll 2 ll 3 ll 0.5 ll 4.5 ll 30 ll

Ligate at RT for 1 h (Prepare PAGE gel during ligation). 3.1.8. PAGE purification of ligated dsDNA products, 62 bp band (1.5 h)  Prepare the glass and spacers (1.5 mm) for pouring the gel.  Prepare a 12% non-denaturing TBE gel in a 50 ml conical vial in the following order.

Reagents

12% (1 gel)

12% (2 gels)

40% Acrylamide stock (ml) 10 TBE (ml) DEPC (ml) (Mol. Biol. Grade)

6 2 To 20 ml

9 3 To 30 ml

 Add 10 ll of 6 GLB (Gel Loading Buffer) to the 50 ll ligated dsDNA products.  Prepare marker: use 5 ll of Promega 10 bp/25 bp (3 lg) ladder with 1 ll of 6 GLB.  Run the gel until good separation of dyes (160 V, 1 h), purple dye should run close to bottom.  After the run, remove gel from glass, stain the gel with ethidium bromide (use 3 ll EtBr in 100 ml DEPC water) by slowly shaking the gel for 10 min.  The ligated products should have a size of 63 bp. (63 bp = 50 adapter (22 bp) + MmeI-digested tag (20 bp) + 30 dsDNA adapter (21 bp))  Prepare 0.5 ml tubes by punching one hole (1 mm diameter) at the bottom of the tube with needle.  Under UV, slice corresponding gel band between 50 bp and 75 bp Promega 25 bp ladder with blade, put into 0.5 ml tube (1 tube per sample) (Fig. 2). BAND IS NOT VISIBLE at this step, so the area of the gel corresponding to the markers 50–75 bp should be cut.  Put the 0.5 ml tube (with gel) into a 2 ml tube and centrifuge for 1 min at 10,000 RPM. Remove the 0.5 ml tube (there should be no gel left in the 0.5 ml tube) and the crushed gel should be in the 2 ml tube. Add 300 ll of nuclease-free water.  Mix by inversion and shake at medium speed (Mix-all lab mixer) over night at RT (6–8 h). Days 2 & 3:

 Add: 180 ll (120 ll for 20 ml gel) of freshly prepared 10% APS and 13.8 ll (9.2 ll for 20 ml gel) of TEMED. Mix well and be careful to avoid aeration of the solution.  Immediately insert the appropriate comb. Allow the acrylamide to polymerize for 30 min at room temperature.  Assemble gel in electrophoresis rig. Add 1 TBE running buffer.

3.1.9. Concentrate the ligated dsDNA products by EtOH precipitation (3 h)  Transfer the suspension (with gel) into a COSTAR Spin-X centrifuge filter (0.5 lM filter).  Spin down max speed RT for 1 min. Discard the filter.  Add 2 ll of GlycoBlue (5 mg/ml) to tube (300 ll), 30 ll of 3 M sodium acetate (NaOAc) and add 900 ll of 100% EtOH.  Mix well by inversion and place at 80 °C for 2 h.  Spin at max speed (13,000 RPM) at 4 °C for 30 min.  Discard supernatant. Wash of the pellet with 70% EtOH and then a quick spin to collect and remove residual supernatant.  Dry the pellet. Dissolve the pellet in 30 ll of nuclease-free water. 3.1.10. Final PCR amplification of PARE library (30 min)

Duplex adapter ligated DNA 100% DMSO 5 GC Buffer 10 mM (each) dNTP mix 30 TruSeq sRNA Index primer 50 Final PCR primer (10 lM) dH2O Phusion polymerase TOTAL

30 ll 1.5 ll 10 ll 1.25 ll 0.5 ll 0.5 ll 5.75 ll 0.5 ll 50 ll

PCR: 98 °C for 30 s, (98 °C for 10 s, 58 °C for 30 s, 72 °C for 20 s) 15 cycles, 72 °C for 10 min, 4 °C HOLD. (Prepare PAGE gel in the meantime, during the PCR) 3.1.11. PAGE purification of the final PCR products, 128 bp band (5 h) Fig. 2. PAGE purification of ligated dsDNA products (63-bp band). The gel fragment was isolated containing DNA between 50 bp and 75 bp, estimated based on the 25bp DNA ladder from Promega.

 Prepare the glass and spacers (1.5 mm) for pouring the gel.

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 Prepare a 6% non-denaturing TBE gel in a 50 ml conical vial in the following order.

 Mix by inversion and shake at medium speed (Mix-all lab mixer) overnight at RT (4+ h). 3.1.12. Concentrate the final library by EtOH precipitation (3 h)

Reagents

6% (1 gel)

6% (2 gels)

40% Acrylamide stock (ml) 10 TBE (ml) DEPC (ml) (Mol. Biol. Grade)

3 2 To 20 ml

4.5 3 To 30 ml

 Add: 180 ll (120 ll for 20 ml gel) of freshly prepared 10% APS and 13.8 ll (9.2 ll for 20 ml gel) of TEMED. Mix well and be careful to avoid aeration of the solution.  Immediately insert the appropriate comb. Allow the acrylamide to polymerize for 30 min at room temperature.  Assemble gel in electrophoresis rig. Add 1 TBE running buffer.  Add 10 ll of 6 GLB to the 50 ll ligated dsDNA products.  Prepare marker: use 5 ll of Promega 10 bp/25 bp (3 lg) ladder with 1 ll of 6 GLB.  Run the gel until good separation of dyes (145 V, 1 h), purple dye should run close to bottom.  After the run, remove gel from glass, stain the gel with ethidium bromide (use 3 ll EtBr in 100 ml DEPC water) by slowly shaking the gel for 10 min.  The ligated products should have a size of 128 bp. (128 bp = 50 adapter (45 bp) + insert (20 bp) + 30 index adapter (63 bp))  Prepare 0.5 ml tubes by punching one hole (1 mm diameter) at the bottom of the tube with needle.  Under UV, there should be a single band apparent near the 125 bp DNA marker; cut out that band and put it into a 0.5 ml tube (1 tube per sample) (Fig. 3).  Put the 0.5 ml tube (with gel) into a 2 ml tube and centrifuge for 1 min at 10,000 RPM. Remove the 0.5 ml tube (there should be no gel left in the 0.5 ml tube); the crushed gel should be in the 2 ml tube. Add 300 ll of nuclease free water.

 Transfer the suspension (with gel) into a COSTAR Spin-X centrifuge filter (0.5 lM filter).  Spin down max speed RT for 1 min. Discard the filter.  Add 2 ll of GlycoBlue (5 mg/ml) to tube (300 ll), 30 ll of 3 M sodium acetate (NaOAc) and add 900 ll of 100% EtOH.  Mix well by inversion and place at 80 °C for 2 h.  Spin at max speed (13,000 RPM) at 4 °C for 30 min.  Discard supernatant. Wash the pellet with 70% EtOH and then perform a quick spin to collect and remove residual supernatant.  Dry the pellet. Dissolve the pellet in 10 ll of nuclease-free water. 3.1.13. Quality assessment of PARE library for Illumina sequencing (30 min)  Determine the fragment size profile of the PARE library by analysis on an Agilent Bioanalyzer High Sensitivity DNA chip. Optimal PARE library should exist as a tight fragment of approximately 130 bp ± 5 bp (Fig. 4).  Determine specific dsDNA concentration of the sample by fluorometry (Qubit High Sensitivity Kit or Picogreen). A high quality PARE library at 10 nM should have a fluorometric concentration reading of approximately 850 pg/ll.  Determine molarity of specific PARE library fragments using the concentration and fragment size profile. Adjust the concentration of the PARE library to 10 nM using Tris–HCl 10 mM, pH 8.5 with 0.1% Tween 20. The addition of 0.1% Tween helps to prevent adsorption of the sample to plastic tubes following freeze–thaw cycles.  PARE libraries with different individual indexes can be pooled together for multiplexing (depends on the size of the genome

Fig. 3. PAGE purification of the final PCR products (128-bp band). The target bands were cut out, at the position around 125 bp, based on the 25 bp DNA ladder from Promega.

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Fig. 4. Determination of fragment size profile of the PARE library by analysis on an Agilent Bioanalyzer High Sensitivity DNA chip. For an optimal PARE library, the tight size range of approximately 130 bp is clearly detected and no secondary peaks are present; the lower marker (35 bp) and upper marker (10,380 bp) are as indicated.

and the desired sequencing depth, for example, eight libraries can be equally pooled for sequencing on Hi-Seq and get 25 million reads for each). The final concentration of individual libraries was adjusted to 10 nM and 10 ll from each library was taken and pooled together. The pooled sample can be directly sent to a sequencing facility with Illumina Hi-Seq machine. Most users STOP here. The following part provides instructions for a sequencing facility to handle PARE libraries on an Illumina HiSeq 2500 machine. 3.2. Sequencing 3.2.1. Preparation of PARE library for cluster generation and HiSeq 2500 sequencing (30 min)  Combine the PARE library and Tris–Cl 10 mM, pH 8.5 in the 0.2 ml tube so that the final concentration of PARE library is 2 nM in 10 ll. For a 10 nM PARE library this would be 2 ll of the PARE library combined with 8 ll Tris–Cl 10 mM, pH 8.5.  Add 10 ll of 0.1 N NaOH to the 0.2 ml tube.  Vortex briefly and pulse centrifuge to collect sample.  Incubate for exactly 5 min at room temperature to denature the PARE library into single strands.  Transfer 20 ll of denatured PARE library to a 1.5 ml tube containing 980 ll of pre-chilled Hybridization Buffer (supplied in TruSeq SR Cluster Kit V3). The final concentration of this solution is 20 pM. Invert several times to mix, pulse centrifuge, and keep on ice.  Further dilute the 20 pM solution to 10 pM in pre-chilled Hybridization Buffer to a total volume of 1000 ll. Invert several times to mix, pulse centrifuge, and keep on ice until ready to load. This dilution is the hybridization fraction and may vary depending on the hybridization characteristics of individual cBot Cluster Generation Systems.

 Dispense 120 ll of each PARE library pool into 1 tube of an 8tube strip. Set aside on ice until ready to load on the cBot.  Dilute the PARE sequencing primer to 500 pM in Hybridization Buffer. Prepare 120 ll PARE sequencing primer for each PARE library in the 8-strip tube.  Dispense 120 ll of PARE sequencing primer into the appropriate tubes of a 8-strip tube, corresponding to the same tubes which the PARE library is loaded from the previous step.  During cluster generation on the cBot, it is necessary to select the ‘‘SR_TubeStripHyb’’ recipe because the PARE sequencing primer is not compatible with the standard Illumina TruSeq Sequencing primer.  Load the template and primer tube strips into the appropriate positions on the cBot. Follow Illumina standard procedures to perform single-read cluster generation with tube-strip primer hybridization.  After cluster generation and primer hybridization, follow Illumina standard procedures to perform a 50-cycle Single-Read SBS run. If PARE samples are multiplexed as pools the standard Illumina Multiplexing Primer is required for the 7-cycle Index Read.

4. Anticipated results A PARE library constructed following this protocol can be finished within 3 days. Different libraries could be pooled with up to 8 libraries in a channel; this should produce 25 million reads per library if analyzed using an Illumina HiSeq instrument. The variation in the number of distinct reads in a library (i.e. different sequences – some people call these ‘‘unique reads’’) is expected, depending on the complexity of the 50 -monophosphorylated ends that exist in a given tissue and organism. Ideally, libraries for miRNA-target RNAs pair identification would have low levels of t/ rRNAs and high complexity, although these factors may be influenced by both technical and biological factors.

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This improved PARE construction method can save time for the process and reduce the cost compared with an earlier protocol [8]. Furthermore, libraries constructed from this protocol are typically of high quality in terms of read number and complexity. Large numbers of libraries can be prepared and pooled for sequencing, using individual indexes. These characteristics make the PARE libraries suitable for the study of the ‘‘degradome’’ in different tissues, treatments, or species.

6. Trouble shooting  Low input: amount of total RNA to start can be as low as 40 lg.  No final product bands can be seen in gel: increase the final PCR cycle.  Low number of sequencing clusters: use fresh Indexed TruSeq 30 PCR primers.  High level of t/rRNA contamination: poor quality of RNA.

5. Comparison with the previous PARE protocol [8] Acknowledgements We updated the primers for reverse transcription and PCR to make the library compatible with the HiSeq sequencing platform from Illumina. The data generated by this new streamlined method are still the same format as the previous protocol (20 nt sequence tags from MmeI digestion), thus won’t affect the analysis step. Compared to the previous protocol, this new method is significantly faster (from 6–7 days to 2–3 days) and consistently produces libraries with high complexity, an important improvement as one major issue with the previous protocol was the occasionally generation of low complexity libraries. We’ve also updated several reagents/kits used in previous protocol to make it easier for handling multiple samples at the same time, including the following:  mRNA isolation: changed from using Oligotex kit (Qiagen) to using DynabeadsÒ mRNA Purification Kit (Invitrogen)  adapter removal: changed from using Microcon columns (Millipore) to AgencourtÒ AMPureÒ XP (Beckman-Coulter)  double-stranded DNA adapter: changed from ‘‘top, 50 -p-TCG TAT GCC GTC TTC TGC TTG-30 ; and bottom, 50 -CAA GCA GAA GAC GGC ATA CGA NN-30 ; p, phosphate group (Integrated DNA Technologies)’’ to ‘‘top: 50 -TGG AAT TCT CGG GTG CCA AGG-30 (PAGE purified), and bottom: 50 -CCT TGG CAC CCG AGA ATT CCA NN-30 (PAGE purified), no modification’’  30 final PCR primer: changed from P7 primer to the Indexed TruSeq 30 PCR primers, Index 1–24.

This work was supported by a grant from the United Soybean Board and the North Central Soybean Research Program, as well as by funding from the Agriculture and Food Research Initiative Competitive Grants Program Grant No. 2012-67013-19396, from the USDA National Institute of Food and Agriculture. We thank Shujun Luo from Illumina for help to design the RNA adapters and primers that are compatible with the HiSeq instrument. References [1] M.J. Axtell, Annu. Rev. Plant Biol. 64 (2013) 137–159. [2] M.W. Rhoades, B.J. Reinhart, L.P. Lim, C.B. Burge, B. Bartel, D.P. Bartel, Cell 110 (2002) 513–520. [3] M.W. Jones-Rhoades, D.P. Bartel, Mol. Cell 14 (2004) 787–799. [4] E. Allen, Z. Xie, A.M. Gustafson, J.C. Carrington, Cell 121 (2005) 207–221. [5] M.A. German, M. Pillay, D.H. Jeong, A. Hetawal, S. Luo, P. Janardhanan, V. Kannan, L.A. Rymarquis, K. Nobuta, R. German, E. De Paoli, C. Lu, G. Schroth, B.C. Meyers, P.J. Green, Nat. Biotechnol. 26 (2008) 941–946. [6] C. Addo-Quaye, T.W. Eshoo, D.P. Bartel, M.J. Axtell, Curr. Biol. 18 (2008) 758– 762. [7] B.D. Gregory, R.C. O’Malley, R. Lister, M.A. Urich, J. Tonti-Filippini, H. Chen, A.H. Millar, J.R. Ecker, Dev. Cell 14 (2008) 854–866. [8] M.A. German, S. Luo, G. Schroth, B.C. Meyers, P.J. Green, Nat. Protoc. 4 (2009) 356–362.